Effect of dissolved oxygen regulation on the content of ganoderic acid S and R in mycelium of Ganoderma lucidum in a two-stage culture by oscillatory-stationary culture

Effect of dissolved oxygen regulation on the content of ganoderic acid S and R in mycelium of Ganoderma lucidum in a two-stage culture by oscillatory-stationary culture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of dissolved oxygen regulation on the content of ganoderic acid S and R in mycelium of Ganoderma lucidum in a two-stage culture by oscillatory-stationary culture Chenmin Tang, Yanfang Liu, Xingyi Jiang, Chuanhong Tang, Jie Feng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3577529/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Currently, liquid fermentation is regarded as a feasible method for cultivating thyme as contrast to subentity culture. Nevertheless, the production of ganoderic acid S and R is still rather limited, thus requiring the need for parameter adjustment in the liquid fermentation process. The influence of thype, an oxygen bacterium, on the viability of microorganisms during liquid fermentation is significant. Moreover, liquid ferments can be classified into two distinct categories: oscillating and static. Prior studies have primarily focused on analyzing these two approaches separately, leading to discrepancies in the consistency and concentration of the fluid form and thyme content. Therefore, the primary objective of this study is to enhance the soluble parameter and incorporate both vibrating and static cultivation methods to facilitate the liquid erection of the grass. The experimental findings indicate that the manipulation of dissolved oxygen during the oscillation stage can significantly enhance the growth of Ganoderma lucidum biomass. However, its impact on the levels of ganoderic acids S and R is comparatively less pronounced. The manipulation of dissolved oxygen during the resting stage yielded contrasting outcomes, leading to a substantial increase in the levels of ganoderic acids S and R, while exerting a lower impact on biomass. The findings of this study demonstrated that the growth of G. lucidum primarily occurred during the oscillating culture stage, while the accumulation of ganoderic acid S and R was predominantly observed during the stationary culture stage. This approach introduces a novel concept for the liquid fermentation of G. lucidum , while simultaneously offering valuable insights for the industrial-scale production of ganoderic acid. Ganoderma lucidum liquid fermentation oscillatory-stationary culture oxygen ganoderic acids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Ganoderma lucidum , a fungus species of great historical importance in China, is recognized for its notable medicinal and nutritional attributes (Wu et al., 2019 ; Tang & Zhong, 2002 ). Numerous studies have shown evidence that G. lucidum possesses a wide range of naturally occurring bioactive chemicals, primarily consisting of Ganoderma triterpenes, polysaccharides, and polyphenols. Ganoderma triterpenoids exhibit notable characteristics such as the suppression of neoplastic cells, modulation of blood lipid levels, and augmentation of immune system activity, among other elements. The primary triterpenoids present in the liquid fermented mycelium of G. lucidum consist of sterols, while the concentration of ganoderic acid is notably little (Jiang et al., 2023 ; Xu & Zhong, 2012 ). Furthermore, the metabolic mechanism of ganoderic acid remains ambiguous (Qian et al., 2013 ; Cai et al., 2020 ), the promotion of ganoderic acid metabolism through molecular biology to regulate the major enzymes in the metabolic pathway remains unattainable. Hence, the augmentation of ganoderic acid concentration may alone be achieved by the optimization of the fermentation process. Nevertheless, the procurement of ganoderic acids is a significant obstacle (Hsu et al., 2008 ; Li et al., 2022 ; Zhao et al., 2019 ). In contrast to substrate cultivation, liquid fermentation has emerged as a viable method for the extraction of ganoderma triterpenes. This is primarily attributed to its shorter cultivation duration, consistent environmental conditions, and enhanced automation capabilities (Fazenda et al., 2008 ). The majority of prior fermentation methods for G. lucidum have been developed using the culture conditions of G. lucidum substrate cultivation or similar fermentation parameters of microorganisms. While these methods have supported the growth of G. lucidum , the metabolite production of G. lucidum during liquid fermentation remains relatively low. Consequently, the full potential of liquid fermentation for G. lucidum has not been fully realized (Oludemi et al., 2018 ; Ye et al., 2018 ). The growth of G. lucidum and the metabolism of its active products are significantly influenced by the dissolved oxygen parameter. This is attributed to the accelerated growth of mycelium and heightened life activities observed during liquid fermentation. The synthesis and metabolism of various products necessitate a substantial amount of oxygen. Therefore, the availability of dissolved oxygen plays a crucial role in facilitating the aforementioned processes (Zhong et al., 2014 ; Xu et al., 2015 ). In the fermentation of strain G0017, the yields of triterpenoids and sterols in the mycelium were enhanced by adjusting the aeration rate in the fermentation broth. By systematically modifying the dissolved oxygen conditions through graded adjustments, the triterpenoid yield reached 3.34 g/L and the sterol yield reached 3.46 g/L. These values represent a significant increase of 69.54% and 75.63%, respectively, compared to the fixed aeration rate of 1.5 L/min (Feng et al., 2021 ); the experimental results demonstrated that the highest levels of biomass and triterpenes production were attained at 8.27 g/L and 171.2 mg/L. Respectively, when the dissolved oxygen levels were manipulated via orthogonal trials during the fermentation process (Yue et al., 2010 ); when the aeration rate during fermentation ranged from 0.3 to 0.55 L/min, the polysaccharide content in G. lucidum mycelium was found to be 68.5 g/kg (Hu, 2006 ).Moreover, the current management of dissolved oxygen is solely implemented during oscillatory culture, with limited regulation of dissolved oxygen in the stationary culture mode. Research has demonstrated that G. lucidum exhibits the formation of a resilient upper layer of mycelium during the process of standing fermentation. This mycelium layer is abundant in G. lucidum triterpenoids (Fang & Zhong, 2002 ; Zhang et al., 2010 ), but the growth of G. lucidum is characterized by poor performance and limited biomass production. To address the above problems, this study aims to combine the two culture methods of G. lucidum , oscillation and resting, and then regulate the dissolved oxygen at different time points in the oscillation and resting phases, respectively. In order to obtain an optimal fermentation strategy that takes into account the biomass and ganoderic acid yield of G. lucidum , and to provide a new idea for the large-scale production of ganoderic acids in G. lucidum . Materials and methods Strain The strain used in this study was G. lucidum G0023, which was provided by Shanghai Edible Mushroom Branch Center of Agricultural Microbiology Center of China Microbial Strain Preservation and Management Committee (CMSMC). The culture was inoculated and incubated ai 26 ℃ for 7 days, then stored at 4 ℃ for 2 mouths. Medium and Culture Conditions Slant medium: Potato dextrose agar (PDA, Becton, Dickinson and Company) was prepared with distilled water in the proportion of 39 g/L and sterilized at 121℃ for 20 min. The recipe for seed medium which was 20 g/L of glucose, 4 g/L of yeast extract, 1.5 g/L of MgSO 4 ·7H 2 O, 1.5 g/L KH 2 PO 4 , and natural pH. Sterilize at 121 ℃ for 30 min and prepare for use. For the seed culture, three pieces of approximately soybean-sized clusters were picked and inoculated in 250 mL triangular flasks with a liquid volume of 100 mL. The culture was incubated at 26 ℃ and 150 r/min for 10 days.The fermenter medium was the same as seed medium. Divided into two groups, a (oscillatory phase control) and b (stationary phase control). The incubation conditions were 26 ℃, 150 r/min for 7 days. The stationary culture conditions were 26 ℃ and incubated for 14 days. (This position for Fig. 1 ) (This position for Fig. 2 ) Analytical Methods Determination of Mycelial Biomass After completion of fermentation, solid-liquid separation was performed by centrifugation at 8000 r/min for 30 min, washed twice, and lyophilized to constant weight (Feng et al., 2014 ; Sudhakar et al., 2021 ). Determination of Total Triterpenes The lyophilized mycelium was extracted with 95% ethanol solution according to the appropriate material-liquid ratio, and the supernatant was centrifuged at 8000 r/min for 30 min after ultrasonication for 2 h. The supernatant was then used for the determination of triterpenoids in G. lucidum . The triterpene content of G. lucidum was determined by the colorimetric method of vanillin-glacial acetic acid (Feng et al., 2015 ; Da et al., 2015 ). High Performance Liquid Chromatography analysis of the ethanol extract of mycelium The mycelium was extracted with anhydrous ethanol at the appropriate material-liquid ratio, sonicated for 1 h, centrifuged at 8000 r/min for 10 min, and the supernatant was passed through a 0.22 µm filter membrane and then detected in the liquid phase. The flow rate was 1.0 mL/min, the column temperature was 30 ℃, the injection volume was 10 µL, and the detection wavelength was set at 240 nm (Guo et al., 2013 ; Liu & Zhong, 2011 ). The sample was separated on YMC-Pack ODS-AQ column (250×4.6 mml. D. S-5µm, 12 nm; Sigma). According to available reports, the mobile phase containing Acetonitrile (A) and 0.5% (v/v) acetic acid (B). The gradient elution program: 0–45 min, 55% A → 75% A; 45–55 min, 75% A→ 85% A; 55–63 min, 85% A → 100% A; 63–80 min, 100% A → 55% A (Wagner et al., 2003 ; Nishibota et al., 1986). The linear regression equation of ganoderic acids S is Y = 15389X-84168 (R2 = 0.9986); The linear regression equation of ganoderic acids R is Y = 1597.6X-31405(R2 = 0.9981). The standard was bought from Sigma. Determination of anti-tumor activity The anti-tumor activity assay method was referring to existing methods with slight modification (Zhang et al., 2010 ; Chen et al., 2001; Cao et al., 2021). 5 mg of ethanol extract of mycelium was weighed and 4 mL of dimethyl sulfoxide was added to configure a sample solution with a final action mass concentration of 6.25 µg/mL, which was then diluted to 3.13 µg/mL and 1.56 µg/mL, respectively. Results and discussion The sample status after fermentation of G. lucidum (This position for Fig. 3 ) As shown in Fig. 3 . The observed phenomenon indicates stratification within the fermentation broth. The upper mycelium exhibits a high degree of density and possesses commendable hardness, whilst the lower mycelium assumes a gelatinous nature. It is possible that the upper mycelium is more prone to air circulation, resulting in a certain level of desiccation of the upper mycelium. The compact structure of the upper mycelium leads to limited evaporation of water from the lower mycelium, hence maintaining high water content in the lower mycelium. Analysis of biomass and ganoderic acid content of G. lucidum when dissolved oxygen is regulated during the oscillatory culture (This position for Fig. 4 ) Glucose utilization is a valid indicator of the growth of G. lucidum (Hsu et al., 2021 ). Nevertheless, due to the presence of stratification in the fermentation broth and its lack of homogeneity, the assessment of G. lucidum growth was limited to biomass evaluation. Figure 4 illustrates that the biomass of G. lucidum after fermentation could be significantly affected by regulating the dissolved oxygen in the shock incubation stage, which was the highest among the biomass in the A4 group, reaching 13.1 g/L, which was 16.9% higher than that of the control. A2 had been in a constant state of closed air, its biomass was significantly lower than others. It's worth noting that the A3 and A4 appear to be significantly different. Based on the observed development curve of G. lucidum fermentation, it is plausible to believe that G. lucidum undergoes an acclimation phase during the pre-fermentation period, characterized by a sluggish growth rate and a limited reliance on oxygen. Following a fermentation period of 3.5 days, G. lucidum enters the logarithmic growth phase characterized by accelerated development and heightened metabolic activity. Consequently, the organism exhibits an increased oxygen requirement. The oxygen conditions given by A4 are highly conducive to the growth of G. lucidum . Consequently, the biomass of A4 will experience a substantial increase. (This position for Fig. 5 ) According to data in Fig. 5 , the content of total triterpenes in different groups has minor elevation compared with the control(A1). Among them, the total triterpene content of A2 was higher, reaching 61.56 mg/g, which was 38.33% higher than the control. Currently, there are relevant reports suggesting that the low oxygen environment can increase the triterpene content of G. lucidum (Milovanovic et al., 2023 ; Zhou et al., 2019 ). Ganoderma triterpenes are lanosterane derivatives that undergo significant oxidation, while G. lucidum contains ergosterol and yeast sterols (Hashim et al., 2016 ). The colorimetric approach used for detecting the triterpene concentration of G. lucidum is susceptible to significant interference. Hence, it is imperative to do additional qualitative and quantitative study of ganoderma triterpenes using High-Performance Liquid Chromatography (HPLC) (Keypour et al., 2009). (This position for Fig. 6 ) Ganoderic acid, a triterpenoid compound, exhibits notable anti-tumor properties and is comparatively more challenging to procure than other triterpenoids, specifically sterols. Hence, the primary aim of optimizing the fermentation process was to enhance the production yield of diverse ganoderic acids (Fang et al., 2002 ). The Fig. 6 has shown that ganoderic acid were detected in the upper layer of the mycelium of all samples and the peak times were close to each other, while no obvious ganoderic acid were detected in the lower layer of mycelium. Based on the qualitative analysis of the standards, it was found that the components with peak times around 56.5 and 66.5 min were ganoderic acid S and R, respectively. The findings of this study demonstrate that the upper mycelium exhibits a significant enrichment of ganoderic acids S and R, whereas the lower mycelium shows negligible levels of these ganoderic acids. Additionally, the majority of sterols discovered by the colorimetric approach are present in the upper mycelium. Hence, solely the quantification of ganoderic acid S and R was conducted in the upper mycelium. (This position for Table 1 ) Table 1 The results of HPLC fingerprints of ganoderic acid S and R in the oscillation phase with regulated dissolved oxygen Sample Ganoderic acid S(mg/g) Ganoderic acid R(mg/g) Total (mg/g) A1 8.7595 ± 0.2536 c 1.9346 ± 0.0407 b 10.6941 ± 0.2132 c A2 9.7170 ± 0.1220 b 1.7713 ± 0.0371 c 12.1852 ± 0.1332 ab A3 9.6746 ± 0.0959 b 1.9945 ± 0.0320 b 11.4459 ± 0.2304 b A4 10.6314 ± 0.2853 a 2.4682 ± 0.0176 a 12.6259 ± 0.1741 a The data in Table 1 has shown that the upper mycelium of all experimental groups was enriched in ganoderic acids S and R, and the total content was slightly higher than that of the control. Among them, the A4 had the most prominent content of ganoderic acid S and R, reaching 10.6314 mg/g and 2.4682 mg/g, which were 21.37% and 27.58% higher than the control, respectively. The findings of this study indicate that the manipulation of dissolved oxygen levels during the oscillating culture phase has a minor impact on the metabolism of ganoderic acid, while exerting a notable favorable influence on the biomass of G. lucidum . Additionally, it has been demonstrated that the presence of upper mycelium in the fermentation medium of G. lucidum has a beneficial impact on the production of S and R ganoderic acids. Currently, the primary factor contributing to the development of the top mycelium appears to be water evaporation. However, subsequent treatment of the lower mycelium under comparable conditions did not result in a significant rise in ganoderic acid content. Hence, it is plausible that factors other than mere water evaporation may contribute to the development of the upper mycelium layer in G. lucidum . There is speculation on the potential influence of a low oxygen environment on the regulation of key enzymes within the ganoderic acid metabolic pathway. This speculation suggests that the regulation of dissolved oxygen levels may contribute to the expression of a specific functional gene (Shiao & Lee, 2005 ). Analysis of biomass and ganoderic acid content of G. lucidum when dissolved oxygen is regulated during the stationary culture (This position for Fig. 7 ) According to the data presented in Fig. 7 , the issolved oxygen was regulated during the stationary incubation phase and the biomass of all three experimental groups was not significantly different from the control. The potential reason for this occurrence could be attributed to the fact that G. lucidum had attained a state of stability and achieved its peak biomass during the oscillation culture phase. Hence, the predominant mycelium growth seen throughout the fermentation process of G. lucidum occurred mostly in the oscillation stage. Consequently, the modulation of dissolved oxygen levels in the stationary stage would not exert a significant influence on the biomass production of G. lucidum . The synthesis of ganoderic acid is hypothesized to predominantly occur during the stationary phase. (This position for Fig. 8 ) It has been shown that G. lucidum is more favorable for the synthesis of ganoderic acid when fermented in a stationary state (Zhang & Zhong, 2010 ). The data shows in Fig. 8 indicates that there was a slight elevation of triterpenoids in all groups of samples after modulation of dissolved oxygen during the stationary incubation phase compared to the control. This result is similar to that of the oscillatory stage modulated dissolved oxygen, which may also be due to the fact that the colorimetric method is susceptible to the interference of sterols. Therefore, the results of colorimetric method are only for reference, and further detection and analysis by HPLC is needed to determine the type and content of ganoderic acid in mycelium. (This position for Fig. 9 ) Figure 9 illustrates that the upper mycelium of each group had distinct peaks of ganoderic acid S and R, while the lower mycelium contained almost no ganoderic acids S and R. This result is similar to that of the oscillatory phase modulation of dissolved oxygen, which still needs to be calculated by the regression equation for its specific content. (This position for Table 2 ) Table 2 The results of HPLC fingerprints of ganoderic acid S and R in the stationary phase with regulated dissolved oxygen Sample Ganoderic acid S(mg/g) Ganoderic acid R(mg/g) Total (mg/g) B1 7.8978 ± 0.3451 c 4.3023 ± 0.3030 b 12.2001 ± 0.2292 c B2 12.0469 ± 0.3556 b 4.3861 ± 0.2459 b 16.6587 ± 0.3673 b B3 11.9594 ± 0.3945 b 5.6324 ± 0.3081 a 16.3454 ± 0.5989 b B4 19.5959 ± 0.3172 a 4.6118 ± 0.3376 ab 25.2283 ± 0.5452 a Based on the regression equation, the content of ganoderic acid S and R was calculated as shown in Table 2 . The contents of ganoderic acid S and R in all experimental groups were more significantly increased, with the highest total amount of ganoderic acid S and R in B4 reaching 25.2283 mg/g, which was 106.79% higher than that of the control. The findings of this study demonstrate that the manipulation of dissolved oxygen levels during the oscillation stage can have a substantial impact on the synthesis of ganoderic acid. Additionally, these results provide evidence that the production of ganoderic acid occurs mostly during the stationary culture stage. The hypothesis posits that the utilization of the stationary culture method may exert a positive regulatory effect on the key enzymes involved in the metabolic pathway of ganoderic acid. However, due to the current lack of understanding regarding the specific metabolic pathway of ganoderic acid, it remains challenging to ascertain the precise key enzyme that experiences a positive influence. Consequently, additional research endeavors are warranted to shed light on this matter. (This position for Fig. 10 ) It has been reported in the literature that ganoderic acids S and R have good anti-tumor activity (Yang et al., 2005 ; Liu et al., 2018 ). Because of ganoderic acid inhibits topoisomerase activity and inhibits DNA synthesis to the extent that it contributes to cancer cell death (Li et al., 2005 ), and ganoderic acid has been found to improve the functionality of the immune system in the human body, specifically by enhancing the activity of natural killer cells (NK cells) and promoting the activity of lymphocytes (Wang et al., 2007 ). Hence, the efficacy of ganoderic acid in terms of its bioactivity was confirmed through the application of the ethanol extract of the upper mycelium in the intervention of two distinct tumor cell lines, namely K562 and L1210. The ethanol extracts of the samples had significant inhibitory effects on both K562 and L1210 cell lines, demonstrating a clear correlation with concentration. At a dosage of 6.25 µg/mL, the inhibition rate for both tumor cells was seen to be approximately 90%. The findings of this study suggest that there is a greater similarity in the types of ganoderic acid present in the two groups of samples. Additionally, the results provide evidence that the ethanolic extract exhibits a high concentration of ganoderic acid. Conclusion Most of recent studies on the enhancement of ganoderic acid by modulation of dissolved oxygen were focused on regulation of dissolved oxygen during the oscillatory phase. In contrast, few studies have been conducted to regulate dissolved oxygen during the stationary phase. More importantly is that oscillatory and stationary are performed independently. Both cultures have different advantages, and if it is possible to combine the benefits of both methods, it may be possible to further promote the synthesis of ganoderic acid. This study shown that the oscillatory-stationary two-stage culture methodology, building upon the foundation of a previous single-stage culture approach, while also independently regulating the dissolved oxygen characteristics of each stage. A comprehensive analysis was conducted to examine the impact of various modulations on the production of ganoderic acids S and R. Additionally, the anticancer efficacy of the ethanol extract derived from fermented mycelium was assessed. The experimental findings demonstrated that the manipulation of dissolved oxygen during the oscillation phase had a significant impact on the growth of G. lucidum and its biomass production. Among the tested conditions, A4 exhibited the highest biomass yield, surpassing the control group by 16.9%. However, the modulation of dissolved oxygen had a limited effect on the concentrations of ganoderic acid S and R. The modulation of dissolved oxygen during the oscillation phase was found to have a significant positive impact on the synthesis of ganoderic acid S and R. Specifically, the highest levels of ganoderic acid S and R were observed in the B4 sample, surpassing the control group. However, the effect on Ling 106.79% was not as pronounced. It is worth noting that this modulation did not lead to an increase in the biomass of G. lucidum . The findings of this investigation provide evidence that the culture stage of Ganoderma biomass accumulation and the synthesis of a significant quantity of ganoderic acid have a clear impact on the concentration of ganoderic acid S and R. The metabolic pathway of ganoderic acid remains uncertain; however, this work provides evidence that the dissolved oxygen parameter is capable of facilitating the synthesis of ganoderic acid S and R. Subsequent investigations may be conducted to further explore the implications of this finding. By manipulating the dissolved oxygen levels during Ganoderma fermentation, it is possible to examine the enzyme activities associated with the metabolic pathway of ganoderic acid. This approach may enable the identification of specific genes influenced by variations in dissolved oxygen, thereby shedding light on the underlying mechanism. The study presents a novel perspective on Ganoderma fermentation and offers valuable insights for the industrial-scale production of ganoderic acid. Declarations Authors Contributions JF designed the research. MCT and XYJ conducted the research and analyzed the data and wrote the manuscript. CHT, YFL and JSZ provided help and suggestions for this study. Funding This work was supported by Agriculture Research System of Shanghai of China ([2022] 9), Project of Shanghai Academy of Agricultural and Sciences Excellent Team (2022A-03), Shanghai Academy of Agricultural Sciences Climbing Project. Ethical Approval This article does not contain any studies with human participants or animals performed Consent to Participate Informed consent was obtained from all individual participants included in the study. Consent to Publish The authors affirm that human research participants provided informed consent for publication Ethics Statement This study does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest All the authors declare that they have no conflict of interest. Data Availability All data generated or analyzed during this study are included in this article References Cai S, Xiao H, Wang X, Lin S, Zhong JJ (2020) Bioconversion of a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid by a crude enzyme from Ganoderma lucidum . Process Biochem 95. https://doi.org/10.1016/j.procbio.2020.05.002 Cao LP, Jin HL, Liang Q, Yang HY, Li SG, Liu ZC, Yuan ZW (2022) A new anti-tumor cytotoxic triterpene from Ganoderma lucidum . 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Food Funct 9(1):209–226. https://doi.org/10.1039/c7fo01601h Qian J, Xu H, Song JY, Xu J, Zhu YJ, Chen SL (2013) Genome-wide analysis of simple sequence repeats in the model medicinal mushroom Ganoderma lucidum . Gene 512(2):331–336. https://doi.org/10.1016/j.gene.2012.09.127 Shiao MS, Lee LW (2005) Polysaccharides and oxygenated triterpenes in the fungus Ganoderma lucidum : Genomic approaches to their biological functions and biosynthesis. 230th National Meeting of the American-Chemical-Society .2005 Sudhakar MP, Ravel M, Perumal K (2021) Pretreatment and process optimization of bioethanol production from spent biomass of Ganoderma lucidum using Saccharomyces cerevisiae. Fuel: A journal of fuel science 306. https://doi.org/10.1016/j.fuel.2021.121680 Tang YJ, Zhong JJ (2002) Fed-batch fermentation of Ganoderma lucidum for hyperproduction of polysaccharide and ganoderic acid. 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Biotechnol Bioeng 111(11):2358–2365. https://doi.org/10.1002/bit.25288 Xu YN, Zhong JJ (2012) Impacts of calcium signal transduction on the fermentation production of antitumor ganoderic acids by medicinal mushroom Ganoderma lucidum . Biotechnol Adv 30(6):1301–1308. https://doi.org/10.1016/j.biotechadv.2011.10.001 Yang HL, Chen GH, Li YQ (2005) A quantum chemical and statistical study of ganoderic acids with cytotoxicity against tumor cell. Eur J Med Chem 40(10):972–976. https://doi.org/10.1016/j.ejmech.2005.04.015 Ye LY, Liu SR, Xie F, Zhao LL, Wu XP, Olaf K (2018) Enhanced production of polysaccharides and triterpenoids in Ganoderma lucidum fruit bodies on induction with signal transduction during the fruiting stage. PLoS ONE 13(4). https://doi.org/10.1371/journal.pone.0196287 Yue QX, Song XY, Ma C, Feng LX, Guan SH, Wu WY, Yang M, Jiang BH, Liu X, Cui YJ, Guo DA (2010) Effects of triterpenes from Ganoderma lucidum on protein expression profile of HeLa cells. Phytomedicine 17(8):606–613. https://doi.org/10.1016/j.phymed.2009.12.013 Zhang JS, Tang QJ, Zhou CY, Silva LD, Nguyen LD, Reutter W, Fan H, H (2010) GLIS, a bioactive proteoglycan fraction from Ganoderma lucidum , displays anti-tumour activity by increasing both humoral and cellular immune response. Life Sci 87:628–637. https://doi.org/10.1016/j.lfs.2010.09.026 Zhang WX, Tang YJ, Zhong JJ (2010) Impact of oxygen level in gaseous phase on gene transcription and ganoderic acid biosynthesis in liquid static cultures of Ganoderma lucidum . Bioprocess Biosyst Eng 33(6):683–690. https://doi.org/10.1007/s00449-009-0379-9 Zhang WX, Zhong JJ (2010) Effect of oxygen concentration in gas phase on sporulation and individual ganoderic acids accumulation in liquid static culture of Ganoderma lucidum . J Biosci Bioeng 109(1):37–40. https://doi.org/10.1016/j.jbiosc.2009.06.024 Zhao C, Fan J, Liu Y, Guo W, Cao H, Xiao J, Wang Y, Liu B (2019) Hepatoprotective activity of Ganoderma lucidum triterpenoids in alcohol-induced liver injury in mice, an iTRAQ-based proteomic analysis. Food Chem 271(1):148–156. https://doi.org/10.1016/j.foodchem.2018.07.115 Zhong JJ, Xu YN, Tan GY, Bai L (2014) Signal transduction engineering: a powerful platform technology for enhancing secondary metabolite production. New Biotechnol 31(2):S23–S24. https://doi.org/10.1016/j.nbt.2014.05.1666 Zhou D, Zhou FX, Ma JF, Ge FH (2019) Microcapsulation of Ganoderma Lucidum spores oil: Evaluation of its fatty acids composition and enhancement of oxidative stability. Ind Crops Prod 131:1–7. https://doi.org/10.1016/j.indcrop.2019.01.031 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3577529","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":247200069,"identity":"c7774d70-bd05-4046-9996-be03640b2ee1","order_by":0,"name":"Chenmin Tang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chenmin","middleName":"","lastName":"Tang","suffix":""},{"id":247200072,"identity":"bcec508f-6598-406c-8350-45d271a9a08c","order_by":1,"name":"Yanfang Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yanfang","middleName":"","lastName":"Liu","suffix":""},{"id":247200074,"identity":"6f65ef57-b598-4c9b-b4ec-fae0ffeb13a4","order_by":2,"name":"Xingyi Jiang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xingyi","middleName":"","lastName":"Jiang","suffix":""},{"id":247200077,"identity":"43da0fc7-7aba-4623-bbd7-6bc629c76c80","order_by":3,"name":"Chuanhong Tang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chuanhong","middleName":"","lastName":"Tang","suffix":""},{"id":247200079,"identity":"3498ec7d-3d0c-426e-8467-14217bd64725","order_by":4,"name":"Jie Feng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYLCCCiBmbGZgfJBQUUOMemYGhjMQLcwGD84cI0ELELBJPmxhJqxBfkb+MYkDFXfsmtuZj1UkNrAx8Ld3J+DVYnAjmU3iwJlnyY3NbGk3EnfIMEicObsBvxaJZDbpj22HkxmbecxuJJ5hA4rk4tciPwNoy8F/IC383woS25gJa2EAOexgw2E7oC1sDERpMTjz2NjiwLHDCYzNbMYSCWeO8RD0i3x74sMbB2oO2xv2H3748UdFjRx/ey8BhwkkgKnEjQ0QPg9+5SDAfwBM2csTVjoKRsEoGAUjFQAAQAVM283EGCIAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Jie","middleName":"","lastName":"Feng","suffix":""},{"id":247200092,"identity":"82f5156a-248d-41e1-9dae-35e2ebd37d01","order_by":5,"name":"Jingsong Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jingsong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-11-08 05:44:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3577529/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3577529/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46239019,"identity":"9e9216be-1958-453e-bf13-4a087c4a9767","added_by":"auto","created_at":"2023-11-10 17:45:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":109169,"visible":true,"origin":"","legend":"\u003cp\u003eDissolved oxygen regulation strategy in oscillation stage (A1: Control; A2: Closed air for 7 days; A3: Ventilation for 3.5 days, closed air for 3.5 days; A4: Closed air for 3.5 days, ventilation for 3.5 days. The same as below)\u003c/p\u003e","description":"","filename":"floatimage114.png","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/1b8dc736bd2ad43720afe5e6.png"},{"id":46239025,"identity":"8ac22898-a3b1-4295-b415-ff52f8f66eda","added_by":"auto","created_at":"2023-11-10 17:45:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99666,"visible":true,"origin":"","legend":"\u003cp\u003eDissolved oxygen regulation strategy at stationary stage (B1: Control; B2: Closed air for 14 days; B3: Ventilation for 7 days, closed air for 7days; B4: Closed air for 7 days, ventilation for 7 days. The same as below)\u003c/p\u003e","description":"","filename":"floatimage217.png","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/ab87fbcaccddde0e3fd5a867.png"},{"id":46239026,"identity":"3ca605e6-345c-465c-a4d4-1ff820d31554","added_by":"auto","created_at":"2023-11-10 17:45:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":132535,"visible":true,"origin":"","legend":"\u003cp\u003eDetails of fermentation broth\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/1d95026cbb8292d41fd6c8e3.png"},{"id":46240273,"identity":"c1343eb9-ae88-4417-b668-7006cef1aeba","added_by":"auto","created_at":"2023-11-10 17:53:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22658,"visible":true,"origin":"","legend":"\u003cp\u003eBiomass of \u003cem\u003eG. lucidum\u003c/em\u003eregulating dissolved oxygen during oscillatory culture (Different lowercase letters indicate the significant differences between different samples (P \u0026lt; 0.05). The same below.)\u003c/p\u003e","description":"","filename":"floatimage411.png","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/774bb2f494c4711744610446.png"},{"id":46240272,"identity":"55fc49f1-8845-40d0-bf99-85679eb1ff58","added_by":"auto","created_at":"2023-11-10 17:53:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":25435,"visible":true,"origin":"","legend":"\u003cp\u003eContent of total triterpenes in \u003cem\u003eG. lucidum\u003c/em\u003e regulating dissolved oxygen during oscillatory culture\u003c/p\u003e","description":"","filename":"floatimage59.png","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/7b07f17b07d680fcb2161a69.png"},{"id":46239020,"identity":"425890b0-046a-4251-939e-ef3302c96ec6","added_by":"auto","created_at":"2023-11-10 17:45:01","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":265112,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC fingerprints of ganoderic acid in the oscillation phase with regulated dissolved oxygen (a: upper mycelium; b: lower mycelium)\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/4a9524b912797a9cfe6b2c1e.jpeg"},{"id":46240274,"identity":"9c5e9f37-9a95-4fc8-978b-9f2a8d7b1c6d","added_by":"auto","created_at":"2023-11-10 17:53:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3998,"visible":true,"origin":"","legend":"\u003cp\u003eBiomass of \u003cem\u003eG. lucidum\u003c/em\u003eregulating dissolved oxygen during stationary culture\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/02acc8a60a73fc0d70702b5e.png"},{"id":46239021,"identity":"5d2253df-eb6b-4521-9a5a-c6155140bf5d","added_by":"auto","created_at":"2023-11-10 17:45:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4415,"visible":true,"origin":"","legend":"\u003cp\u003eContent of total triterpenes in \u003cem\u003eG. lucidum\u003c/em\u003e regulating dissolved oxygen during stationary culture\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/f528c8ac3e91f4e2cdaab9ad.png"},{"id":46239027,"identity":"dd327484-d11c-48fe-a49d-ddbf91720190","added_by":"auto","created_at":"2023-11-10 17:45:01","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":263062,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC fingerprints of ganoderic acid in the stationary phase with regulated dissolved oxygen (a: upper mycelium; b: lower mycelium)\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/1952af23666f8f9df98caf76.jpeg"},{"id":46239028,"identity":"3a85ec0f-136a-4397-98c5-e1a7ab7287ef","added_by":"auto","created_at":"2023-11-10 17:45:01","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":835670,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro anti-tumour activity analysis of ethanolic extracts from the upper mycelium of \u003cem\u003eG. lucidum\u003c/em\u003efermentations with two-stage oscillation-stationary regulation of dissolved oxygen (a: K562 cells; b: L1210 cells)\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/97b7b170a1dffbf723be3772.jpeg"},{"id":50848323,"identity":"b29391a5-9c7d-4f4e-bed8-fb081994b755","added_by":"auto","created_at":"2024-02-08 10:10:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1102883,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3577529/v1/b8c32db1-716a-4fc3-be6d-481d71a45496.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of dissolved oxygen regulation on the content of ganoderic acid S and R in mycelium of Ganoderma lucidum in a two-stage culture by oscillatory-stationary culture","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eGanoderma lucidum\u003c/em\u003e, a fungus species of great historical importance in China, is recognized for its notable medicinal and nutritional attributes (Wu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tang \u0026amp; Zhong, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Numerous studies have shown evidence that \u003cem\u003eG. lucidum\u003c/em\u003e possesses a wide range of naturally occurring bioactive chemicals, primarily consisting of Ganoderma triterpenes, polysaccharides, and polyphenols. Ganoderma triterpenoids exhibit notable characteristics such as the suppression of neoplastic cells, modulation of blood lipid levels, and augmentation of immune system activity, among other elements. The primary triterpenoids present in the liquid fermented mycelium of \u003cem\u003eG. lucidum\u003c/em\u003e consist of sterols, while the concentration of ganoderic acid is notably little (Jiang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu \u0026amp; Zhong, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, the metabolic mechanism of ganoderic acid remains ambiguous (Qian et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Cai et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the promotion of ganoderic acid metabolism through molecular biology to regulate the major enzymes in the metabolic pathway remains unattainable. Hence, the augmentation of ganoderic acid concentration may alone be achieved by the optimization of the fermentation process. Nevertheless, the procurement of ganoderic acids is a significant obstacle (Hsu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast to substrate cultivation, liquid fermentation has emerged as a viable method for the extraction of ganoderma triterpenes. This is primarily attributed to its shorter cultivation duration, consistent environmental conditions, and enhanced automation capabilities (Fazenda et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The majority of prior fermentation methods for \u003cem\u003eG. lucidum\u003c/em\u003e have been developed using the culture conditions of \u003cem\u003eG. lucidum\u003c/em\u003e substrate cultivation or similar fermentation parameters of microorganisms. While these methods have supported the growth of \u003cem\u003eG. lucidum\u003c/em\u003e, the metabolite production of \u003cem\u003eG. lucidum\u003c/em\u003e during liquid fermentation remains relatively low. Consequently, the full potential of liquid fermentation for \u003cem\u003eG. lucidum\u003c/em\u003e has not been fully realized (Oludemi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ye et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe growth of \u003cem\u003eG. lucidum\u003c/em\u003e and the metabolism of its active products are significantly influenced by the dissolved oxygen parameter. This is attributed to the accelerated growth of mycelium and heightened life activities observed during liquid fermentation. The synthesis and metabolism of various products necessitate a substantial amount of oxygen. Therefore, the availability of dissolved oxygen plays a crucial role in facilitating the aforementioned processes (Zhong et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the fermentation of strain G0017, the yields of triterpenoids and sterols in the mycelium were enhanced by adjusting the aeration rate in the fermentation broth. By systematically modifying the dissolved oxygen conditions through graded adjustments, the triterpenoid yield reached 3.34 g/L and the sterol yield reached 3.46 g/L. These values represent a significant increase of 69.54% and 75.63%, respectively, compared to the fixed aeration rate of 1.5 L/min (Feng et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); the experimental results demonstrated that the highest levels of biomass and triterpenes production were attained at 8.27 g/L and 171.2 mg/L. Respectively, when the dissolved oxygen levels were manipulated via orthogonal trials during the fermentation process (Yue et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e); when the aeration rate during fermentation ranged from 0.3 to 0.55 L/min, the polysaccharide content in \u003cem\u003eG. lucidum\u003c/em\u003e mycelium was found to be 68.5 g/kg (Hu, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).Moreover, the current management of dissolved oxygen is solely implemented during oscillatory culture, with limited regulation of dissolved oxygen in the stationary culture mode. Research has demonstrated that \u003cem\u003eG. lucidum\u003c/em\u003e exhibits the formation of a resilient upper layer of mycelium during the process of standing fermentation. This mycelium layer is abundant in \u003cem\u003eG. lucidum\u003c/em\u003e triterpenoids (Fang \u0026amp; Zhong, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), but the growth of \u003cem\u003eG. lucidum\u003c/em\u003e is characterized by poor performance and limited biomass production.\u003c/p\u003e \u003cp\u003eTo address the above problems, this study aims to combine the two culture methods of \u003cem\u003eG. lucidum\u003c/em\u003e, oscillation and resting, and then regulate the dissolved oxygen at different time points in the oscillation and resting phases, respectively. In order to obtain an optimal fermentation strategy that takes into account the biomass and ganoderic acid yield of \u003cem\u003eG. lucidum\u003c/em\u003e, and to provide a new idea for the large-scale production of ganoderic acids in \u003cem\u003eG. lucidum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrain\u003c/h2\u003e \u003cp\u003eThe strain used in this study was \u003cem\u003eG. lucidum\u003c/em\u003e G0023, which was provided by Shanghai Edible Mushroom Branch Center of Agricultural Microbiology Center of China Microbial Strain Preservation and Management Committee (CMSMC). The culture was inoculated and incubated ai 26 ℃ for 7 days, then stored at 4 ℃ for 2 mouths.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMedium and Culture Conditions\u003c/h2\u003e \u003cp\u003eSlant medium: Potato dextrose agar (PDA, Becton, Dickinson and Company) was prepared with distilled water in the proportion of 39 g/L and sterilized at 121℃ for 20 min. The recipe for seed medium which was 20 g/L of glucose, 4 g/L of yeast extract, 1.5 g/L of MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 1.5 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and natural pH. Sterilize at 121 ℃ for 30 min and prepare for use. For the seed culture, three pieces of approximately soybean-sized clusters were picked and inoculated in 250 mL triangular flasks with a liquid volume of 100 mL. The culture was incubated at 26 ℃ and 150 r/min for 10 days.The fermenter medium was the same as seed medium. Divided into two groups, a (oscillatory phase control) and b (stationary phase control). The incubation conditions were 26 ℃, 150 r/min for 7 days. The stationary culture conditions were 26 ℃ and incubated for 14 days.\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical Methods\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of Mycelial Biomass\u003c/h2\u003e \u003cp\u003eAfter completion of fermentation, solid-liquid separation was performed by centrifugation at 8000 r/min for 30 min, washed twice, and lyophilized to constant weight (Feng et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sudhakar et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Total Triterpenes\u003c/h2\u003e \u003cp\u003eThe lyophilized mycelium was extracted with 95% ethanol solution according to the appropriate material-liquid ratio, and the supernatant was centrifuged at 8000 r/min for 30 min after ultrasonication for 2 h. The supernatant was then used for the determination of triterpenoids in \u003cem\u003eG. lucidum\u003c/em\u003e. The triterpene content of \u003cem\u003eG. lucidum\u003c/em\u003e was determined by the colorimetric method of vanillin-glacial acetic acid (Feng et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Da et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHigh Performance Liquid Chromatography analysis of the ethanol extract of mycelium\u003c/h2\u003e \u003cp\u003eThe mycelium was extracted with anhydrous ethanol at the appropriate material-liquid ratio, sonicated for 1 h, centrifuged at 8000 r/min for 10 min, and the supernatant was passed through a 0.22 \u0026micro;m filter membrane and then detected in the liquid phase. The flow rate was 1.0 mL/min, the column temperature was 30 ℃, the injection volume was 10 \u0026micro;L, and the detection wavelength was set at 240 nm (Guo et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Liu \u0026amp; Zhong, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The sample was separated on YMC-Pack ODS-AQ column (250\u0026times;4.6 mml. D. S-5\u0026micro;m, 12 nm; Sigma). According to available reports, the mobile phase containing Acetonitrile (A) and 0.5% (v/v) acetic acid (B). The gradient elution program: 0\u0026ndash;45 min, 55% A \u0026rarr; 75% A; 45\u0026ndash;55 min, 75% A\u0026rarr; 85% A; 55\u0026ndash;63 min, 85% A \u0026rarr; 100% A; 63\u0026ndash;80 min, 100% A \u0026rarr; 55% A (Wagner et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Nishibota et al., 1986). The linear regression equation of ganoderic acids S is Y\u0026thinsp;=\u0026thinsp;15389X-84168 (R2\u0026thinsp;=\u0026thinsp;0.9986); The linear regression equation of ganoderic acids R is Y\u0026thinsp;=\u0026thinsp;1597.6X-31405(R2\u0026thinsp;=\u0026thinsp;0.9981). The standard was bought from Sigma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of anti-tumor activity\u003c/h2\u003e \u003cp\u003eThe anti-tumor activity assay method was referring to existing methods with slight modification (Zhang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chen et al., 2001; Cao et al., 2021). 5 mg of ethanol extract of mycelium was weighed and 4 mL of dimethyl sulfoxide was added to configure a sample solution with a final action mass concentration of 6.25 \u0026micro;g/mL, which was then diluted to 3.13 \u0026micro;g/mL and 1.56 \u0026micro;g/mL, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eThe sample status after fermentation of\u003c/b\u003e \u003cb\u003eG. lucidum\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The observed phenomenon indicates stratification within the fermentation broth. The upper mycelium exhibits a high degree of density and possesses commendable hardness, whilst the lower mycelium assumes a gelatinous nature. It is possible that the upper mycelium is more prone to air circulation, resulting in a certain level of desiccation of the upper mycelium. The compact structure of the upper mycelium leads to limited evaporation of water from the lower mycelium, hence maintaining high water content in the lower mycelium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of biomass and ganoderic acid content of\u003c/b\u003e \u003cb\u003eG. lucidum\u003c/b\u003e \u003cb\u003ewhen dissolved oxygen is regulated during the oscillatory culture\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGlucose utilization is a valid indicator of the growth of \u003cem\u003eG. lucidum\u003c/em\u003e (Hsu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Nevertheless, due to the presence of stratification in the fermentation broth and its lack of homogeneity, the assessment of \u003cem\u003eG. lucidum\u003c/em\u003e growth was limited to biomass evaluation. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates that the biomass of \u003cem\u003eG. lucidum\u003c/em\u003e after fermentation could be significantly affected by regulating the dissolved oxygen in the shock incubation stage, which was the highest among the biomass in the A4 group, reaching 13.1 g/L, which was 16.9% higher than that of the control. A2 had been in a constant state of closed air, its biomass was significantly lower than others. It's worth noting that the A3 and A4 appear to be significantly different. Based on the observed development curve of \u003cem\u003eG. lucidum\u003c/em\u003e fermentation, it is plausible to believe that \u003cem\u003eG. lucidum\u003c/em\u003e undergoes an acclimation phase during the pre-fermentation period, characterized by a sluggish growth rate and a limited reliance on oxygen. Following a fermentation period of 3.5 days, \u003cem\u003eG. lucidum\u003c/em\u003e enters the logarithmic growth phase characterized by accelerated development and heightened metabolic activity. Consequently, the organism exhibits an increased oxygen requirement. The oxygen conditions given by A4 are highly conducive to the growth of \u003cem\u003eG. lucidum\u003c/em\u003e. Consequently, the biomass of A4 will experience a substantial increase.\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to data in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the content of total triterpenes in different groups has minor elevation compared with the control(A1). Among them, the total triterpene content of A2 was higher, reaching 61.56 mg/g, which was 38.33% higher than the control. Currently, there are relevant reports suggesting that the low oxygen environment can increase the triterpene content of \u003cem\u003eG. lucidum\u003c/em\u003e (Milovanovic et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ganoderma triterpenes are lanosterane derivatives that undergo significant oxidation, while \u003cem\u003eG. lucidum\u003c/em\u003e contains ergosterol and yeast sterols (Hashim et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The colorimetric approach used for detecting the triterpene concentration of \u003cem\u003eG. lucidum\u003c/em\u003e is susceptible to significant interference. Hence, it is imperative to do additional qualitative and quantitative study of ganoderma triterpenes using High-Performance Liquid Chromatography (HPLC) (Keypour et al., 2009).\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGanoderic acid, a triterpenoid compound, exhibits notable anti-tumor properties and is comparatively more challenging to procure than other triterpenoids, specifically sterols. Hence, the primary aim of optimizing the fermentation process was to enhance the production yield of diverse ganoderic acids (Fang et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e has shown that ganoderic acid were detected in the upper layer of the mycelium of all samples and the peak times were close to each other, while no obvious ganoderic acid were detected in the lower layer of mycelium. Based on the qualitative analysis of the standards, it was found that the components with peak times around 56.5 and 66.5 min were ganoderic acid S and R, respectively. The findings of this study demonstrate that the upper mycelium exhibits a significant enrichment of ganoderic acids S and R, whereas the lower mycelium shows negligible levels of these ganoderic acids. Additionally, the majority of sterols discovered by the colorimetric approach are present in the upper mycelium. Hence, solely the quantification of ganoderic acid S and R was conducted in the upper mycelium.\u003c/p\u003e \u003cp\u003e(This position for Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of HPLC fingerprints of ganoderic acid S and R in the oscillation phase with regulated dissolved oxygen\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGanoderic acid S(mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGanoderic acid R(mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal (mg/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.7595\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2536\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.9346\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0407\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.6941\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2132\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.7170\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1220\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7713\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0371\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.1852\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1332\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.6746\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0959\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.9945\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0320\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.4459\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2304\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.6314\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2853\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.4682\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0176\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.6259\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1741\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe data in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e has shown that the upper mycelium of all experimental groups was enriched in ganoderic acids S and R, and the total content was slightly higher than that of the control. Among them, the A4 had the most prominent content of ganoderic acid S and R, reaching 10.6314 mg/g and 2.4682 mg/g, which were 21.37% and 27.58% higher than the control, respectively. The findings of this study indicate that the manipulation of dissolved oxygen levels during the oscillating culture phase has a minor impact on the metabolism of ganoderic acid, while exerting a notable favorable influence on the biomass of \u003cem\u003eG. lucidum\u003c/em\u003e. Additionally, it has been demonstrated that the presence of upper mycelium in the fermentation medium of \u003cem\u003eG. lucidum\u003c/em\u003e has a beneficial impact on the production of S and R ganoderic acids. Currently, the primary factor contributing to the development of the top mycelium appears to be water evaporation. However, subsequent treatment of the lower mycelium under comparable conditions did not result in a significant rise in ganoderic acid content. Hence, it is plausible that factors other than mere water evaporation may contribute to the development of the upper mycelium layer in \u003cem\u003eG. lucidum\u003c/em\u003e. There is speculation on the potential influence of a low oxygen environment on the regulation of key enzymes within the ganoderic acid metabolic pathway. This speculation suggests that the regulation of dissolved oxygen levels may contribute to the expression of a specific functional gene (Shiao \u0026amp; Lee, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of biomass and ganoderic acid content of\u003c/b\u003e \u003cb\u003eG. lucidum\u003c/b\u003e \u003cb\u003ewhen dissolved oxygen is regulated during the stationary culture\u003c/b\u003e\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the issolved oxygen was regulated during the stationary incubation phase and the biomass of all three experimental groups was not significantly different from the control. The potential reason for this occurrence could be attributed to the fact that \u003cem\u003eG. lucidum\u003c/em\u003e had attained a state of stability and achieved its peak biomass during the oscillation culture phase. Hence, the predominant mycelium growth seen throughout the fermentation process of \u003cem\u003eG. lucidum\u003c/em\u003e occurred mostly in the oscillation stage. Consequently, the modulation of dissolved oxygen levels in the stationary stage would not exert a significant influence on the biomass production of \u003cem\u003eG. lucidum\u003c/em\u003e. The synthesis of ganoderic acid is hypothesized to predominantly occur during the stationary phase.\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt has been shown that \u003cem\u003eG. lucidum\u003c/em\u003e is more favorable for the synthesis of ganoderic acid when fermented in a stationary state (Zhang \u0026amp; Zhong, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The data shows in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e indicates that there was a slight elevation of triterpenoids in all groups of samples after modulation of dissolved oxygen during the stationary incubation phase compared to the control. This result is similar to that of the oscillatory stage modulated dissolved oxygen, which may also be due to the fact that the colorimetric method is susceptible to the interference of sterols. Therefore, the results of colorimetric method are only for reference, and further detection and analysis by HPLC is needed to determine the type and content of ganoderic acid in mycelium.\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates that the upper mycelium of each group had distinct peaks of ganoderic acid S and R, while the lower mycelium contained almost no ganoderic acids S and R. This result is similar to that of the oscillatory phase modulation of dissolved oxygen, which still needs to be calculated by the regression equation for its specific content.\u003c/p\u003e \u003cp\u003e(This position for Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of HPLC fingerprints of ganoderic acid S and R in the stationary phase with regulated dissolved oxygen\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGanoderic acid S(mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGanoderic acid R(mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal (mg/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.8978\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3451\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.3023\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3030\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.2001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2292\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.0469\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3556\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.3861\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2459\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.6587\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3673\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.9594\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3945\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.6324\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3081\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.3454\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5989\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.5959\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3172\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.6118\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3376\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.2283\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5452\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on the regression equation, the content of ganoderic acid S and R was calculated as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The contents of ganoderic acid S and R in all experimental groups were more significantly increased, with the highest total amount of ganoderic acid S and R in B4 reaching 25.2283 mg/g, which was 106.79% higher than that of the control. The findings of this study demonstrate that the manipulation of dissolved oxygen levels during the oscillation stage can have a substantial impact on the synthesis of ganoderic acid. Additionally, these results provide evidence that the production of ganoderic acid occurs mostly during the stationary culture stage. The hypothesis posits that the utilization of the stationary culture method may exert a positive regulatory effect on the key enzymes involved in the metabolic pathway of ganoderic acid. However, due to the current lack of understanding regarding the specific metabolic pathway of ganoderic acid, it remains challenging to ascertain the precise key enzyme that experiences a positive influence. Consequently, additional research endeavors are warranted to shed light on this matter.\u003c/p\u003e \u003cp\u003e(This position for Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt has been reported in the literature that ganoderic acids S and R have good anti-tumor activity (Yang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Because of ganoderic acid inhibits topoisomerase activity and inhibits DNA synthesis to the extent that it contributes to cancer cell death (Li et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and ganoderic acid has been found to improve the functionality of the immune system in the human body, specifically by enhancing the activity of natural killer cells (NK cells) and promoting the activity of lymphocytes (Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Hence, the efficacy of ganoderic acid in terms of its bioactivity was confirmed through the application of the ethanol extract of the upper mycelium in the intervention of two distinct tumor cell lines, namely K562 and L1210. The ethanol extracts of the samples had significant inhibitory effects on both K562 and L1210 cell lines, demonstrating a clear correlation with concentration. At a dosage of 6.25 \u0026micro;g/mL, the inhibition rate for both tumor cells was seen to be approximately 90%. The findings of this study suggest that there is a greater similarity in the types of ganoderic acid present in the two groups of samples. Additionally, the results provide evidence that the ethanolic extract exhibits a high concentration of ganoderic acid.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMost of recent studies on the enhancement of ganoderic acid by modulation of dissolved oxygen were focused on regulation of dissolved oxygen during the oscillatory phase. In contrast, few studies have been conducted to regulate dissolved oxygen during the stationary phase. More importantly is that oscillatory and stationary are performed independently. Both cultures have different advantages, and if it is possible to combine the benefits of both methods, it may be possible to further promote the synthesis of ganoderic acid. This study shown that the oscillatory-stationary two-stage culture methodology, building upon the foundation of a previous single-stage culture approach, while also independently regulating the dissolved oxygen characteristics of each stage. A comprehensive analysis was conducted to examine the impact of various modulations on the production of ganoderic acids S and R. Additionally, the anticancer efficacy of the ethanol extract derived from fermented mycelium was assessed. The experimental findings demonstrated that the manipulation of dissolved oxygen during the oscillation phase had a significant impact on the growth of \u003cem\u003eG. lucidum\u003c/em\u003e and its biomass production. Among the tested conditions, A4 exhibited the highest biomass yield, surpassing the control group by 16.9%. However, the modulation of dissolved oxygen had a limited effect on the concentrations of ganoderic acid S and R. The modulation of dissolved oxygen during the oscillation phase was found to have a significant positive impact on the synthesis of ganoderic acid S and R. Specifically, the highest levels of ganoderic acid S and R were observed in the B4 sample, surpassing the control group. However, the effect on Ling 106.79% was not as pronounced. It is worth noting that this modulation did not lead to an increase in the biomass of \u003cem\u003eG. lucidum\u003c/em\u003e. The findings of this investigation provide evidence that the culture stage of Ganoderma biomass accumulation and the synthesis of a significant quantity of ganoderic acid have a clear impact on the concentration of ganoderic acid S and R. The metabolic pathway of ganoderic acid remains uncertain; however, this work provides evidence that the dissolved oxygen parameter is capable of facilitating the synthesis of ganoderic acid S and R. Subsequent investigations may be conducted to further explore the implications of this finding. By manipulating the dissolved oxygen levels during Ganoderma fermentation, it is possible to examine the enzyme activities associated with the metabolic pathway of ganoderic acid. This approach may enable the identification of specific genes influenced by variations in dissolved oxygen, thereby shedding light on the underlying mechanism. The study presents a novel perspective on Ganoderma fermentation and offers valuable insights for the industrial-scale production of ganoderic acid.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJF designed the research. MCT and XYJ conducted the research and analyzed the data and wrote the manuscript. CHT, YFL and JSZ provided help and suggestions for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Agriculture Research System of Shanghai of China ([2022] 9), Project of Shanghai Academy of Agricultural and Sciences Excellent Team (2022A-03), Shanghai Academy of Agricultural Sciences Climbing Project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that human research participants provided informed consent for publication\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCai S, Xiao H, Wang X, Lin S, Zhong JJ (2020) Bioconversion of a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid by a crude enzyme from \u003cem\u003eGanoderma lucidum\u003c/em\u003e. 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Ind Crops Prod 131:1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.indcrop.2019.01.031\u003c/span\u003e\u003cspan address=\"10.1016/j.indcrop.2019.01.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ganoderma lucidum, liquid fermentation, oscillatory-stationary culture, oxygen, ganoderic acids","lastPublishedDoi":"10.21203/rs.3.rs-3577529/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3577529/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrently, liquid fermentation is regarded as a feasible method for cultivating thyme as contrast to subentity culture. Nevertheless, the production of ganoderic acid S and R is still rather limited, thus requiring the need for parameter adjustment in the liquid fermentation process. The influence of thype, an oxygen bacterium, on the viability of microorganisms during liquid fermentation is significant. Moreover, liquid ferments can be classified into two distinct categories: oscillating and static. Prior studies have primarily focused on analyzing these two approaches separately, leading to discrepancies in the consistency and concentration of the fluid form and thyme content. Therefore, the primary objective of this study is to enhance the soluble parameter and incorporate both vibrating and static cultivation methods to facilitate the liquid erection of the grass. The experimental findings indicate that the manipulation of dissolved oxygen during the oscillation stage can significantly enhance the growth of \u003cem\u003eGanoderma lucidum\u003c/em\u003e biomass. However, its impact on the levels of ganoderic acids S and R is comparatively less pronounced. The manipulation of dissolved oxygen during the resting stage yielded contrasting outcomes, leading to a substantial increase in the levels of ganoderic acids S and R, while exerting a lower impact on biomass. The findings of this study demonstrated that the growth of \u003cem\u003eG. lucidum\u003c/em\u003e primarily occurred during the oscillating culture stage, while the accumulation of ganoderic acid S and R was predominantly observed during the stationary culture stage. This approach introduces a novel concept for the liquid fermentation of \u003cem\u003eG. lucidum\u003c/em\u003e, while simultaneously offering valuable insights for the industrial-scale production of ganoderic acid.\u003c/p\u003e","manuscriptTitle":"Effect of dissolved oxygen regulation on the content of ganoderic acid S and R in mycelium of Ganoderma lucidum in a two-stage culture by oscillatory-stationary culture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-10 17:44:56","doi":"10.21203/rs.3.rs-3577529/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd65956d-1ba9-46ce-8fc1-ffd9227d50c3","owner":[],"postedDate":"November 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-08T10:02:08+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-10 17:44:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3577529","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3577529","identity":"rs-3577529","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}